the myristoylated amino-terminus of an arabidopsis calcium
TRANSCRIPT
The myristoylated amino-terminus of an Arabidopsis calcium-dependent protein kinase mediates plasma membrane localization
Sheen X. Lu • Estelle M. Hrabak
Received: 16 July 2012 / Accepted: 15 April 2013 / Published online: 23 April 2013
� The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract Calcium-dependent protein kinases (CDPK)
are a major group of calcium-stimulated kinases found in
plants and some protists. Many CDPKs are membrane-
associated, presumably because of lipid modifications at
their amino termini. We investigated the subcellular loca-
tion and myristoylation of AtCPK5, a member of the
Arabidopsis CDPK family. Most AtCPK5 was associated
with the plasma membrane as demonstrated by two-phase
fractionation of plant microsomes and by in vivo detection
of AtCPK5-GFP fusion proteins. AtCPK5 was a substrate
for plant N-myristoyltransferase and myristoylation was
prevented by converting the glycine at the proposed site of
myristate attachment to alanine (G2A). In transgenic
plants, a G2A mutation completely abolished AtCPK5
membrane association, indicating that myristoylation was
essential for membrane binding. The first sixteen amino
acids of AtCPK5 were sufficient to direct plasma mem-
brane localization. In addition, differentially phosphory-
lated forms of AtCPK5 were detected both in planta and
after expression of AtCPK5 in a cell-free plant extract. Our
results demonstrate that AtCPK5 is myristoylated at its
amino terminus and that myristoylation is required for
membrane binding.
Keywords Myristoylation � Acylation � Calcium �Kinase � Membrane targeting � Subcellular localization
Introduction
Calcium is a ubiquitous signaling molecule in plants
(Hashimoto and Kudla 2011). Transient cytosolic calcium
signals are generated in response to environmental signals
or stresses such as light, cold, pathogens, or drought, as
well as in response to internal stimuli, and these signals are
decoded by various calcium-binding proteins (DeFalco
et al. 2010). Since calcium has limited diffusion potential
in the cytosol (Allbritton et al. 1992), rapid and efficient
calcium detection occurs when calcium sensor proteins are
located at cell membranes in close proximity to sites of
calcium influx.
Proteins in the calcium-dependent protein kinase (CDPK)
family have characteristics that enable them to be effective
transducers of cytosolic calcium fluxes. All CDPKs have a
typical serine/threonine kinase catalytic domain fused via a
regulatory autoinhibitory domain to a calmodulin-like
domain that directly binds calcium (Cheng et al. 2002;
Hrabak et al. 2003; Mehlmer et al. 2010). Calcium binding to
the calmodulin-like domain induces intramolecular confor-
mational changes that lead to rapid and calcium-specific
activation of the catalytic domain (Huang et al. 1996).
CDPKs also contain a highly divergent amino-terminal
variable domain that is critical for correct subcellular
localization of the enzyme (Martin and Busconi 2000; Lu
and Hrabak 2002; Dammann et al. 2003; Raices et al. 2003;
Chehab et al. 2004; Gargantini et al. 2006) and may also play
a role in substrate recognition (Ito et al. 2010).
Although not found in metazoans, CDPKs have been
identified in all plants examined to date, including mosses,
liverworts, green algae, gymnosperms and angiosperms,
and are also found in the apicomplexan protists (Zhang and
Choi 2001). Most plants encode multiple CDPK isoforms.
For example, 34 genes encoding CDPKs have been
S. X. Lu � E. M. Hrabak (&)
Department of Molecular, Cellular & Biomedical Sciences,
University of New Hampshire, Durham, NH 03824, USA
e-mail: [email protected]
Present Address:S. X. Lu
Department of Molecular, Cellular and Developmental Biology,
University of California, Los Angeles, CA 09905, USA
123
Plant Mol Biol (2013) 82:267–278
DOI 10.1007/s11103-013-0061-0
identified in the Arabidopsis thaliana genome (Hrabak
et al. 2003), while there are 29 in rice (Asano et al. 2005)
and at least 21 in Medicago truncatula (Gargantini et al.
2006). CDPK isoforms may vary in substrate specificity,
enzyme kinetics, expression pattern, or subcellular location
(Hrabak et al. 2003; Harper et al. 2004) and are involved in
processes such as carbon and nitrogen metabolism
(Douglas et al. 1998; Asano et al. 2002), plant growth and
development (Ivashuta et al. 2005; Gargantini et al. 2006;
Yoon et al. 2006), defense against pathogens (Romeis et al.
2001; Freymark et al. 2007; Kobayashi et al. 2007), and
responses to hormones and abiotic stresses (Abbasi et al.
2004; Ludwig et al. 2005; Szczegielniak et al. 2005; Ma
and Wu 2007; Zhu et al. 2007; Franz et al. 2011).
Many CDPKs are membrane associated although they
do not contain recognizable transmembrane domains. In
Arabidopsis, 10 of the 34 CDPKs have been localized to
the plasma membrane, peroxisome, or endoplasmic retic-
ulum, while two are predominantly cytosolic (Lu and
Hrabak 2002; Dammann et al. 2003; Rodriguez Milla et al.
2006; Zhu et al. 2007; Coca and San Segundo 2010;
Mehlmer et al. 2010). Membrane binding of CDPKs is
likely mediated by acylation of the amino-terminal variable
domain. Myristoylation was first demonstrated for a zuc-
chini CDPK (Ellard-Ivey et al. 1999) and has subsequently
been reported for CDPKs from other species. In Arabid-
opsis, the variable domain of AtCPK2 is myristoylated and
this modification is required for membrane association (Lu
and Hrabak 2002). Similar results have been reported for
CDPKs from rice (Martin and Busconi 2000), ice plant
(Chehab et al. 2004), potato (Raices et al. 2001; Raices
et al. 2003), and tomato (Rutschmann et al. 2002).
Many myristoylated proteins are known or are predicted
to be involved in cellular signaling pathways (Boisson
et al. 2003; Maurer-Stroh et al. 2004; Resh 2004), and
myristoylation is often required for correct protein func-
tion. For example, in Arabidopsis, myristoylation of the
SOS3 calcium-binding protein is required for salt tolerance
(Ishitani et al. 2000), BON1/CPN1 myristoylation is
required for normal plant growth (Li et al. 2010), and
SnRK1 myristoylation affects the catalytic activity of this
kinase and its role in shoot meristem development (Pierre
et al. 2007).
Protein myristoylation is catalyzed by myristoyl-
CoA:protein N-myristoyltransferase (NMT; Rajala et al.
2000; Farazi et al. 2001). Following removal of the initiator
methionine, NMT catalyzes the formation of a stable amide
bond between myristate (C14:0) and the exposed amino-
terminal glycine (Gly-2) residue of a substrate protein. In
addition to an absolute requirement for Gly-2, the amino
acids immediately following Gly-2 play critical roles in
determining whether a protein can be a substrate for NMT
(Towler et al. 1988; Rocque et al. 1993; Utsumi et al. 2004).
Computer algorithms developed to predict protein myris-
toylation (Maurer-Stroh et al. 2002b; Bologna et al. 2004;
Podell and Gribskov 2004) indicate that many CDPKs have
putative N-myristoylation sites at their amino termini.
In this study, we investigate protein modifications and
subcellular location of a CDPK from Arabidopsis thaliana,
AtCPK5. We demonstrate that AtCPK5 is myristoylated
in vitro, although this was not predicted by all of the
available myristoylation programs, and that the majority of
the AtCPK5 protein in plant cells is associated with the
plasma membrane. We also present evidence that phos-
phorylation of AtCPK5 occurs in plant extracts. Mutation
of Gly-2 prevents myristoylation and abolishes membrane
association of AtCPK5 in plants. Finally, we demonstrate
that the first 16 amino acids of AtCPK5 are sufficient to
direct plasma membrane targeting.
Experimental procedures
Plasmid constructs
The pCPK5-16aa-GUS construct contained a 1,417 bp
genomic DNA fragment followed in-frame by the b-glu-
curonidase (GUS) coding sequence from the E. coli uidA
gene and the nos terminator. The 1,417 bp Arabidopsis
genomic DNA fragment (Arabidopsis gene At4g35310)
consisted of 50 nucleotides of coding sequence preceded
by the 449 bp CPK5 untranslated leader (containing a
224 bp intron) and 918 bp of non-transcribed sequence,
presumed to contain the promoter region. The GUS coding
sequence and the nos terminator were from pBI101
(Clontech, Mountain View, CA, USA). For plant trans-
formation, this entire region was cloned into pBIN19
(Bevan 1984) to create pCPK5-16aa-GUS. The first 16
amino acids of AtCPK5 are MGNSCRGSFKDKLDEG.
Mutagenesis of the glycine codon (GGC) at position 2 to
alanine (GCC) was performed with the QuikChange Site-
Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA)
according to the manufacturer’s instructions to create
pCPK5-G2A-GUS. The presence of the G2A mutation was
confirmed by DNA sequencing. For constructs pCPK5-
16aa-GFP and pCPK5-G2A-GFP, the GUS coding
sequence in pCPK5-16aa-GUS and pCPK5-G2A-GUS was
replaced with the coding sequence for soluble-modified
red-shifted green fluorescent protein (smRS-GFP, Davis
and Vierstra 1998).
Plant transformation and growth conditions
Arabidopsis thaliana (ecotype Columbia) plants were
transformed by the floral dip method (Clough and Bent
1998) and transgenics were selected on solidified
268 Plant Mol Biol (2013) 82:267–278
123
Murashige and Skoog basal medium with Gamborg’s B-5
vitamins (Sigma, St. Louis, MO, USA) and 0.1 % (w/v)
sucrose, pH 5.7, containing 50 mg/L kanamycin. Kana-
mycin-resistant plants were confirmed to contain the
transgene using a rapid PCR method (Klimyuk et al. 1993).
Membrane isolation and aqueous two-phase
partitioning
Seeds from transgenic plants were surface-sterilized and
grown in liquid Murashige and Skoog basal medium with
Gamborg’s B-5 vitamins and 1 % (w/v) sucrose, pH 5.7, at
21 �C with an 18 h photoperiod. Aeration was maintained
on a rotary shaker at 120 rpm. Microsomal membranes
were prepared using a modification of a previously
described procedure (Schaller and DeWitt 1995). All
homogenization and fractionation steps were conducted on
ice or in a cold room with prechilled buffers and equip-
ment. All buffers contained protease inhibitor cocktail
(Roche, Indianapolis, IN, USA). Two-week-old plants
were ground in homogenization buffer (50 mM Tris–HCl,
pH 8.2, 20 % [v/v] glycerol, 2 mM EDTA; 1 ml/g fresh
weight) with a mortar and pestle. The homogenates were
cleared by filtration through Miracloth (Calbiochem, La
Jolla, CA, USA) followed by centrifugation at 5,0009g for
5 min. Microsomal membranes were isolated by centrifu-
gation of the supernatant for 30 min at 125,0009g. Mem-
brane pellets were resuspended in SPK buffer (0.33 M
sucrose, 5 mM KPO4, and 3 mM KCl, pH 7.8) using a
ground glass homogenizer. Aqueous two-phase partitioning
to enrich for plasma membrane vesicles was conducted
essentially as described (Larsson et al. 1987). Resuspended
microsomes were added to a 6.3 % (w/w) DextranT500/
PEG3350 phase mixture prepared in SPK buffer and the
phases were separated at 1,0009g for 5–10 min. Both
upper and lower phases were repartitioned twice with fresh
lower or upper phase, respectively. The final upper and
lower phases were diluted separately in 10 mM Tris–HCl
(pH 7.0) containing 1 mM EDTA and 1 mM EGTA.
Phase-partitioned membranes were collected by centrifu-
gation at 125,0009g for 30 min and resuspended in equal
volumes of SPK buffer.
AtCPK5 antibodies
AtCPK5 rabbit polyclonal antiserum was raised against a
fusion protein containing the first 50 amino acids of
AtCPK5 fused to the carboxyl terminus of glutathione
S-transferase. The construct was made in pGEX-KT
(Hakes and Dixon 1992). Recombinant protein expressed
in E. coli was purified on a glutathione-agarose matrix
(Amersham Bioscience, Piscataway, NJ, USA) and injec-
ted into female New Zealand White rabbits. Crude serum
was precipitated with 50 % ammonium sulfate and redis-
solved in phosphate-buffered saline (PBS) [137 mM NaCl,
4.3 mM Na2HPO4.7H2O, 1.5 mM KH2PO4 2.7 mM KCl].
An affinity matrix for purifying AtCPK5 antibodies was
prepared by cloning the AtCPK5 cDNA into pRSET-A
(Invitrogen, Carlsbad, CA, USA), purifying the expressed
proteins on His-Select nickel affinity gel (Sigma), and
crosslinking the purified AtCPK5-6His to Affi-Gel 15 (Bio-
Rad Laboratories, Hercules, CA, USA). After binding of
the partially purified antibody to this matrix, AtCPK5-
specific antibodies were eluted with 0.1 M glycine (pH 2.7)
and immediately neutralized with 0.1 volume of 1 M Tris–
HCl (pH 8.0), followed by dialysis against PBS. Antibody
was used at a 1000-fold dilution.
Immunoblot analysis
For SDS-PAGE, samples were mixed with 39 Blue
Loading Buffer (New England Biolabs, Beverly, MA,
USA) and incubated at 37 �C for 30 min. After electro-
phoresis at 4 �C, proteins were electroblotted to PVDF
membrane (Millipore, Bedford, MA, USA) and immuno-
detection was performed as described previously (Lu and
Hrabak 2002). Antibodies for detection of specific mem-
branes were: anti-H?-ATPase (DeWitt and Sussman 1995)
for plasma membrane (1:10,000), anti-BiP (Hofte et al.
1992) for endoplasmic reticulum (1:1,000), and anti-b–
ATPase D (Luethy et al. 1993) for mitochondrial mem-
branes (1:100). Membranes were stripped between detec-
tions following the manufacturer’s instructions. Thylakoid
membranes were identified by spectrophotometric detec-
tion of chlorophyll a and b (Lichtenthaler 1987).
In vitro myristoylation assay
AtCPK5 cDNA was inserted into pBluescript II KS
(Stratagene) and expressed under control of the vector’s T7
promoter. The transcript produced from this clone contains
38 bp of sequence from the vector and 18 bp of the
AtCPK5 leader preceding the AtCPK5 translation initiation
codon. There is no alternate start codon in the upstream
sequence. Myristoylation assays were performed with the
TnT Coupled Transcription/Translation Wheat Germ
Extract System (Promega Corp., Madison, WI, USA)
according to the manufacturer’s instructions. cDNAs
encoding either wildtype or mutant AtCPK5 proteins were
in vitro transcribed and translated in the presence of either
10 lCi of L-[35S]-methionine (1,000 Ci/mmol; Amer-
sham) or 50 lCi of [9,10-3H]-myristic acid (54 Ci/mmol;
Amersham Bioscience). Before use, the myristic acid was
dried under nitrogen and resuspended by vortexing in
DEPC-treated water at a concentration of 10 lCi/ll.
Control reactions contained no plasmid. After 90 min
Plant Mol Biol (2013) 82:267–278 269
123
incubation at 30 �C, reaction products were separated by
SDS-PAGE and analyzed by fluorography with Entensify
Universal Autoradiography Enhancer (PerkinElmer, Shel-
ton, CT, USA).
Dephosphorylation assay
Products from in vitro myristoylation assays (10 ll) or the
upper phase from two phase partitioning (10 ll) were
incubated with 5 units of calf intestinal alkaline phospha-
tase (Roche) for 15 min at 30 �C. Control reactions con-
tained phosphatase inhibitor cocktail II (Sigma). The
reactions were subsequently boiled in the presence of 39
Blue Loading Buffer, separated by SDS-PAGE, and
AtCPK5 was visualized by immunoblotting.
b-Glucuronidase assays
b-Glucuronidase (GUS) activity was determined with a
fluorometric assay (Gallagher 1992). For CPK5-16aa-GUS
plants, total membrane-bound GUS activity (measured in
nmol min-1 mg-1) ranged from 1.1 9 104 to 1.8 9 104
(n = 7), while GUS activity from CPK5-G2A-GUS plants
ranged from 1.0 9 103 to 2.4 9 103 (n = 6).
Transient expression of AtCPK5-GFP fusion proteins
p35SBSYFP (Katiyar-Agarwal et al. 2006), pCPK5-16aa-
GFP, or pCPK5-G2A-GFP were purified using a QIAprep
Spin Miniprep Kit (Qiagen Inc., Valencia, CA, USA).
Rosette leaves (0.8–1.2 cm in length) were collected from
3- to 4-week-old Arabidopsis plants and bombarded with
0.8 lg plasmid DNA coated on 480 ug of gold particles
(1 lm; Sigma) using a PDS-1000/He delivery system (Bio-
Rad). The distance between the stop screen and the leaves
was *9 cm and a helium pressure of 650 psi was
employed. Bombarded leaves were incubated in water at
room temperature for 4–5 h. Images were collected using a
SP2 confocal microscope (Leica Microsystems, Inc., Ban-
nockburn, IL, USA), analyzed using MetaMorph 4.5 soft-
ware (Universal Imaging Corp., Downingtown, PA, USA),
and processed with Photoshop (Adobe Systems Inc., San
Jose, CA, USA).
Results
AtCPK5 is associated with the plasma membrane
Several Arabidopsis CDPKs are known to be membrane
bound (Lu and Hrabak 2002; Dammann et al. 2003; Choi
et al. 2005; Coca and San Segundo 2010; Mehlmer et al.
2010) although AtCPK5 has not been studied previously.
To determine whether AtCPK5 was associated with the
plasma membrane or with intracellular membranes, aque-
ous two-phase partitioning was used to fractionate micro-
somes from wildtype plants. This technique enriches
plasma membrane vesicles in the upper phase while other
intracellular membranes partition to the lower phase
(Larsson et al. 1987). After phase separation, both phases
were analyzed by immunoblotting to detect AtCPK5 pro-
tein and protein markers for specific membranes (Fig. 1).
AtCPK5 was highly enriched in the upper phase, similar to
the H?-ATPase plasma membrane marker. As expected,
intracellular membrane marker proteins were enriched in
the lower phase. These results indicate that AtCPK5 is
associated with the plasma membrane but does not exclude
the possibility that some AtCPK5 protein may be located in
the cytosol.
AtCPK5 is myristoylated in vitro
To investigate whether the amino terminus of AtCPK5
contains a myristoylation consensus sequence, two myris-
toylation prediction programs were queried: Myristoylator
(http://us.expasy.org/tools/myristoylator/myristoylator-ref.
html; Bologna et al. 2004) and NMT Predictor (http://
mendel.imp.ac.at/myriatate/SUPLpredictor.htm; Maurer-
Stroh et al. 2002a). Myristoylator predicted myristoylation
Fig. 1 AtCPK5 is localized to the plasma membrane. Membranes
from wildtype Arabidopsis plants were fractionated by aqueous two-
phase partitioning. Equal proportions of the upper and lower phases
were separated by SDS-PAGE, transferred to PVDF membranes, and
analyzed sequentially using antibodies specific for AtCPK5 and for
membrane marker proteins. U upper phase (enriched for plasma
membrane); L lower phase (enriched for intracellular membranes)
270 Plant Mol Biol (2013) 82:267–278
123
of AtCPK5 while NMT Predictor did not. To determine
whether AtCPK5 could be myristoylated in vitro, we used
a cell-free transcription/translation system from wheat
germ that is known to contain NMT activity (Ellard-Ivey
et al. 1999; Lu and Hrabak 2002). Both wildtype AtCPK5
protein and its glycine-2 to alanine (G2A) mutant were
tested. Reactions included either [35S]-methionine for
detection of total protein synthesis or [3H]-myristic acid
for detection of myristoylated proteins. For both DNA
templates, the major product of the [35S]-methionine-
labeled reaction was a protein doublet near the predicted
mass of 62.1 kDa (Fig. 2a). [3H]-myristate was incorpo-
rated into a 62 kDa product when the wildtype AtCPK5
protein was expressed but not when template DNA con-
taining the G2A mutation was used (Fig. 2b). The identity
of these radiolabeled proteins as AtCPK5 was confirmed
by immunoblot analysis with AtCPK5 antibody, which
recognizes the unique amino terminus of AtCPK5
(Fig. 2c). The 50 kDa protein recognized by the AtCPK5
antibodies represents non-specific binding to a wheat germ
protein since it was present in a control reaction that
contained no DNA template (Fig. 2c, lane 1). The smaller,
non-myristoylated proteins routinely observed in
[35S]methionine-labeled reactions may be alternative
translation products from the AtCPK5 constructs or
AtCPK5 degradation products (Fig. 2a). These results
indicate that the AtCPK5 protein can be myristoylated
in vitro and that Gly-2 is essential for myristoylation.
AtCPK5 is phosphorylated in plants and in wheat germ
extract
We noticed that AtCPK5 was often detected as a doublet of
approximately 60 and 62 kDa whether the protein source
was Arabidopsis plant extracts (Fig. 1) or wheat germ
expression extracts (Fig. 2). This doublet was observed for
both wildtype AtCPK5 and the G2A mutant protein, indi-
cating that the second band is not due to the effects of
myristoylation. Phosphorylation can retard the migration of
proteins, so we examined whether the 62 kDa species
represented a phosphorylated form of AtCPK5. Three dif-
ferent protein samples were tested: (1) the upper phase
after aqueous two-phase partitioning of wildtype plant
extracts, (2) wheat germ transcription/translation reactions
expressing wildtype AtCPK5, or (3) wheat germ tran-
scription/translation reactions expressing the G2A-mutated
AtCPK5. Protein samples were treated with alkaline
phosphatase in the presence or absence of alkaline phos-
phatase inhibitor. Alkaline phosphatase treatment resulted
in the disappearance of the 62 kDa species (Fig. 3) and an
increase in abundance of the 60 kDa species. In the pres-
ence of both alkaline phosphatase and alkaline phosphatase
Fig. 2 AtCPK5 is myristoylated in vitro and a G2A mutation
prevents myristoylation. In a wheat germ coupled transcription and
translation system, AtCPK5 protein (lane 2) was expressed in the
presence of either a [35S]methionine; b [3H]myristic acid; or c no
radiolabel, followed by SDS-PAGE and fluorography (a and b) or
immunoblotting (c). Parallel experiments were performed on AtCPK5
protein containing a glycine-2 to alanine mutation (lane 3). Negative
control reactions (lane 1) contained no plasmid template for gene
expression. Arrows indicate AtCPK5 protein
Fig. 3 AtCPK5 is phosphorylated when expressed in planta or
in vitro. The upper phase from two-phase partitioning of Arabidopsis
membranes (left), cell-free wheat germ extract following in vitro
expression of AtCPK5 (center), or cell-free wheat germ extract
following expression of the AtCPK5-G2A mutant protein (right) were
incubated with calf intestinal alkaline phosphatase (AP) and alkaline
phosphatase inhibitor (API). The reaction products were separated by
SDS-PAGE and AtCPK5 was detected by immunoblotting with
AtCPK5 antibody
Plant Mol Biol (2013) 82:267–278 271
123
inhibitor, both the 60 and 62 kDa species were detected.
Slight variations in migration are likely due to differences
in buffer composition of the samples. These results indicate
that phosphorylated AtCPK5 is present in plant extracts,
that phosphorylation also occurs during in vitro synthesis
of AtCPK5 in a cell-free extract, and that myristoylation
and membrane association are not required for phosphor-
ylation of AtCPK5.
The first 16 amino acids of AtCPK5 are sufficient
for plasma membrane localization
Correct subcellular targeting of acylated proteins from
metazoans and yeast often requires only a short region at
the amino-terminus of the protein (Di Paolo et al. 1997;
Gillen et al. 1998; Alsheimer et al. 2000). Previously, we
demonstrated that the amino terminus of a different Ara-
bidopsis CDPK, AtCPK2, was sufficient for its correct
targeting to the endoplasmic reticulum (Lu and Hrabak
2002). To determine whether the amino terminal region of
AtCPK5 was sufficient for its membrane targeting, trans-
genic plants were produced that expressed the first 16
amino acids of AtCPK5 fused to the b-glucuronidase
reporter protein (AtCPK5-16aa-GUS) under control of the
native AtCPK5 promoter. For comparison, plants were
transformed with the T-DNA from pBI121 in which the
GUS protein is expressed from the 35S promoter. To
determine the relative distribution of GUS protein between
cytosol and membranes, plant extracts were centrifuged to
separate microsomes from soluble proteins as described
previously and GUS activity in these two fractions was
measured with a sensitive fluorometric assay. As expected,
in control plants transformed with T-DNA from pBI121,
the majority of the GUS protein was soluble and only a
small amount (2–3 %) was membrane associated, probably
due to non-specific membrane binding or trapping of the
abundant GUS protein in membrane vesicles during prep-
aration of extracts. In AtCPK5-16aa-GUS plant extracts,
the majority (78 ± 3 %) of the GUS enzyme activity was
in the microsomal fraction, indicating that most of the
AtCPK5-GUS fusion protein was membrane associated
and that the first 16 amino acids of AtCPK5 are sufficient
for membrane targeting.
To determine whether the CPK5-16aa-GUS fusion
protein was targeted to the plasma membrane similar to
wildtype AtCPK5 (Fig. 1), the microsomal fraction was
used for aqueous two-phase partitioning. Both native At-
CPK5 protein and the plasma membrane marker were
enriched in the upper phase, while the markers for organ-
ellar membranes were concentrated in the lower phase
(Fig. 4), confirming that the cellular membranes parti-
tioned correctly. The location of the hybrid AtCPK5-16aa-
GUS fusion protein was determined with a fluorometric
assay for GUS activity. The plasma membrane-enriched
upper phase contained more GUS activity per milliliter
than the lower phase (Fig. 4). When expressed as specific
activity to correct for the six-fold greater protein content in
the lower phase, the difference between the amount of
AtCPK5-16aa-GUS fusion protein in the two phases is
apparent. These results indicate that the first 16 amino
acids of AtCPK5 are sufficient to correctly target the sol-
uble GUS protein to the plasma membrane.
The G2A mutation abolishes AtCPK5 membrane
association in plants
To address the importance of AtCPK5 myristoylation in
membrane binding in plants, we examined the effect of
mutation of the myristoylation site on membrane associa-
tion. Site-directed mutagenesis was used to convert the
Fig. 4 The first sixteen amino acids of AtCPK5 are sufficient for
plasma membrane localization. Microsomal membranes from CPK5-
16aa-GUS transgenic plants (expressing GUS protein preceded by the
first 16 amino acids of AtCPK5) were analyzed by aqueous two-phase
partitioning. Equal proportions of the upper and lower phases were
separated by SDS-PAGE and assayed by immunoblotting with
specific antibodies. Chlorophyll absorbance (ng/ml) was measured
spectrophotometrically. The GUS enzyme was assayed fluorometri-
cally and is expressed as activity (lmol/min/ml) and as specific
activity (lmol/min/mg protein). U upper phase, L lower phase
272 Plant Mol Biol (2013) 82:267–278
123
second codon of pCPK5-16aa-GUS from glycine to alanine
(G2A) to create pCPK5-G2A-GUS. Extracts from trans-
genic plants expressing the CPK5-G2A-GUS fusion pro-
tein were separated into soluble and microsomal fractions
by ultracentrifugation. GUS enzyme activity was measured
in both fractions using the fluorometric assay. The majority
(97 %) of the GUS activity was detected in the soluble
fraction, compared with 22 % when plants expressed the
CPK5-16aa-GUS transgene without the G2A mutation.
These results demonstrate that myristoylation is critical for
the membrane binding of AtCPK5 in vivo.
GFP fusions confirm plasma membrane localization
of AtCPK5
To confirm the plasma membrane localization of AtCPK5,
a construct containing the first 16 amino acids of AtCPK5
fused to green fluorescent protein was used for transient
expression in Arabidopsis. pCPK5-16aa-GFP was bom-
barded into Arabidopsis leaves and expression in epidermal
cells was examined by confocal microscopy (Fig. 5). The
quantitative GUS assay data presented earlier indicated that
*80 % of AtCPK5 was plasma membrane associated and
it is evident from a single optical section near the midpoint
of the cell that CPK5-16aa-GFP was found at the cell
periphery, corresponding to plasma membrane-localized
AtCPK5 (Fig. 5d). A maximum intensity projection indi-
cates that fluorescence was also observed in the cytosol and
nucleus, likely corresponding to soluble CPK5-16aa-GFP
fusion protein (Fig. 5c). Fluorescence in nuclei was
observed routinely for all transgenic plants and was not
unexpected since the sizes of free GFP (26.8 kDa) and
CPK5-16aa-GFP (28.5 kDa) are below the exclusion limit
of the nuclear pore complex (Grebenok et al. 1997). In cells
expressing the non-myristoylated CPK5-G2A-GFP fusion
protein, fluorescence was localized primarily in the cytosol
and nucleus (Fig. 5e, f), in a pattern similar to free GFP
(Fig. 5a, b). These data confirm that a substantial propor-
tion of AtCPK5 is localized to the plasma membrane and
that the amino-terminal glycine residue is required for
membrane binding.
Discussion
Calcium-dependent protein kinases have been found in all
plant genomes and function in many cellular processes,
including signaling in response to biotic and abiotic stres-
ses and regulation of carbon and nitrogen metabolism
(Klimecka and Muszynska 2007). In this study, we inves-
tigated the myristoylation and subcellular localization of
AtCPK5 and documented the presence of phosphorylated
forms of this enzyme.
In our experiments, AtCPK5 was routinely detected as a
protein doublet on immunoblots using isoform-specific anti-
bodies. The doublet was also apparent in samples containing
non-myristoylated AtCPK5 and thus the change in elec-
trophoretic behavior was not attributable to acylation.
Phosphorylation can affect protein migration during electro-
phoresis (Peck 2006) and decreased mobility had previously
been documented for a phosphorylated tobacco CDPK
(Romeis et al. 2000; Romeis et al. 2001). Treatment of Ara-
bidopsis protein extracts with alkaline phosphatase caused the
disappearance of the slower-migrating form of AtCPK5,
indicative of phosphorylation. In vitro calcium-dependent
autophosphorylation of AtCPK5 had been documented pre-
viously (Hrabak et al. 1996) and two autophosphorylated
peptides were identified (Hegeman et al. 2006). In these
Fig. 5 Plasma membrane localization of AtCPK5 requires glycine-2.
Arabidopsis epidermal cells transiently expressing various GFP
constructs were observed by confocal microscopy. a and b GFP
control; c and d CPK5-16aa-GFP; e and f CPK5-G2A-GFP. a, c, and
e are projections of 10, 30 and 26 optical sections, respectively; b, d,
and f are single optical sections
Plant Mol Biol (2013) 82:267–278 273
123
experiments, we cannot distinguish autophosphorylation from
phosphorylation catalyzed by other kinases present in Ara-
bidopsis or in the wheat germ extract. Some CDPKs, like
tobacco NtCDPK2, are phosphorylated at specific sites in a
membrane-dependent manner (Witte et al. 2010); however,
phosphorylated forms of AtCPK5 were observed for both
membrane-bound native AtCPK5 and non-myristoylated
AtCPK5-G2A, indicating that phosphorylation was not
dependent upon membrane association.
Calcium-dependent protein phosphorylation has been
reported previously in plasma membrane-enriched plant
cell extracts (Schaller et al. 1992; Verhey et al. 1993;
Baizabal-Aguirre and de la Vara 1997; Iwata et al. 1998)
and some CDPKs are known to localize to the plasma
membrane (Rutschmann et al. 2002; Dammann et al. 2003;
Raices et al. 2003; Chehab et al. 2004; Gargantini et al.
2006; Mehlmer et al. 2010; Kobayashi et al. 2012). We
began our studies of AtCPK5 localization by fractionating
Arabidopsis extracts to separate plasma membranes from
intracellular membranes. Both AtCPK5 and a known
plasma membrane marker were enriched in the upper
fraction while a minor percentage of AtCPK5 was found in
the lower cytosolic fraction, indicating that the membrane-
bound AtCPK5 was primarily associated with the plasma
membrane. The plasma membrane localization of AtCPK5
could facilitate activation of the enzyme by localized cal-
cium signals generated in response to abiotic or biotic
stimuli (reviewed in Kudla et al. 2010). Several plasma
membrane-localized proteins, such as the H?-ATPase and
several ion channels (Pei et al. 1996; Li et al. 1998; Rut-
schmann et al. 2002; Mori et al. 2006; Geiger et al. 2010),
are phosphorylated in a calcium-dependent manner by
CDPKs and represent potential substrates for the mem-
brane-bound form of AtCPK5.
Membrane binding of CDPKs is proposed to be medi-
ated by acylation. This study focused on N-myristoylation,
an irreversible modification that occurs exclusively at an
amino-terminal glycine residue (Rajala et al. 2000). The
enzymology and substrate specificity of myristoyltransfe-
rases from fungi and animals have been characterized
extensively (Towler et al. 1988; Rocque et al. 1993;
Johnson et al. 1994; Lodge et al. 1994; Raju et al. 1995;
Farazi et al. 2001; Utsumi et al. 2004). Plant N-myri-
stoyltransferases are less well understood and differences
between the fungal, human and plant enzymes have been
noted previously (Rocque et al. 1993; Qi et al. 2000;
Boisson et al. 2003; Dumonceaux et al. 2004; Pierre et al.
2007). Algorithms for myristoylation prediction such as
NMT Predictor (http://mendel.imp.univie.ac.at/myristate/;
Maurer-Stroh et al. 2002a, 2002b) and Myristoylator (http://
us.expasy.org/tools/myristoylator/; Bologna et al. 2004)
have been developed primarily using myristoylation data
from fungal and animal NMTs. We have found that these
algorithms do not always work well for predicting plant
myristoylation sites (S. Lu, A. Argyros, and E. Hrabak,
unpublished observations). NMT Predictor rejected AtCPK5
(amino terminal sequence: MGNSCRGSF) as an NMT
substrate, most likely because of the arginine residue at
position 6, which is rarely found in myristoylated proteins
from fungi and mammals (Utsumi et al. 2004). Although
peptides with arginine-6 were not myristoylated in vitro in a
rabbit reticulocyte lysate (Utsumi et al. 2004), our results
using wheat germ lysate clearly demonstrated that AtCPK5
was a bona fide substrate for plant NMT. Boisson et al.
(2003) examined the substrate specificity of one of the two
Arabidopsis N-myristoyltransferases in more detail and
concluded that plant NMTs have relaxed substrate speci-
ficity compared to other enzymes. Subsequently, a Plant-
Specific Myristoylation Predictor (http://plantsp.genomics.
purdue.edu/myrist.html) was developed by Podell and
Gribskov (2004) using only plant myristoylation data.
Because this program was trained using our unpublished
results, this website accurately predicts myristoylation of
Arabidopsis CDPKs, including AtCPK5.
The majority of known CDPKs, including those from
Arabidopsis and rice (Cheng et al. 2002; Hrabak et al.
2003; Ye et al. 2009), contain predicted amino-terminal
myristoylation sites, although myristoylation has been
demonstrated experimentally for only a small group of
CDPKs. Known myristoylated CDPKs are: Arabidopsis
AtCPK2, AtCPK3, AtCPK6, AtCPK9, and AtCPK13 (Lu
and Hrabak 2002; Boisson et al. 2003; Benetka et al. 2008;
Mehlmer et al. 2010), rice OSCPK2 (Martin and Busconi
2000), tomato LeCPK1 (Rutschmann et al. 2002), potato
StCDPK1 (Raices et al. 2001), Mesembryanthemum
McCPK1(Chehab et al. 2004), and Cucurbita CpCPK1
(Ellard-Ivey et al. 1999). Here, we showed that AtCPK5 is
myristoylated on its amino-terminal glycine residue and
localized to the plasma membrane. The first 16 amino acids
of AtCPK5, which contain the myristoylation consensus
sequence, were sufficient to direct plasma membrane tar-
geting of both GUS and GFP. A G2A mutation abolished
both myristoylation and in planta membrane association of
AtCPK5, demonstrating that myristoylation was absolutely
required for membrane binding. Plasma membrane locali-
zation has been demonstrated for other CDPKs, including
AtCPK9, AtCPK13, LeCPK1, StCDPK1 and StCDPK5
(Rutschmann et al. 2002; Raices et al. 2003; Kobayashi
et al. 2012) but other membrane locations are possible.
Myristoylated AtCPK2 is targeted to the endoplasmic
reticulum (Lu and Hrabak 2002), AtCPK3 is nuclear and
perinuclear (Dammann et al. 2003; Mehlmer et al. 2010),
and McCPK1 localizes to multiple membranes (Chehab
et al. 2004). Thus the amino terminal region of different
CDPKs could be used for as targeting signals to direct
proteins to specific membranes.
274 Plant Mol Biol (2013) 82:267–278
123
Myristoylation is unlikely to be the sole determinant of
CDPK membrane binding because the hydrophobicity of
myristate is not sufficient to maintain long-term membrane
association (Peitzsch and McLaughlin 1993; McLaughlin
and Aderem 1995). The first 16 amino acids of AtCPK5
include a potential palmitoylation site at cysteine-5. Pre-
liminary experiments indicate that Cys-5 is palmitoylated
in planta (A. Argyros & E. Hrabak, unpublished data).
Palmitate has about tenfold greater membrane binding
affinity than myristate and palmitoylated proteins are
invariably membrane associated (Resh 2004). The current
model for dual acylation, the kinetic bilayer trapping
hypothesis of Shahinian and Silvius (1995), posits that the
weak hydrophobicity provided by myristate allows proteins
to transiently sample a variety of cellular membranes
where specific palmitoyl transferases reside. Conversely,
mutation of the myristoylation site prevents transient
membrane binding and subsequent cysteine palmitoylation
(Degtyarev et al. 1994; Hallak et al. 1994; Wilson and
Bourne 1995; Martin and Busconi 2001). Thus, even
though wildtype AtCPK5 may be myristoylated and
palmitoylated and the palmitoylation site is still present in
the G2A mutant, we predicted complete loss of AtCPK5
membrane association when myristoylation was prevented,
which is what we observed.
Our results show that AtCPK5 is plasma membrane
localized and that the amino terminus of the protein is
required for correct localization. Myristoylation of the
amino terminal glycine residue of AtCPK5 was critical for
membrane binding. The subcellular location of AtCPK5
will be useful for deciphering its substrates and role in
plant signal transduction pathways.
Acknowledgments We thank Tracy Lambert for technical assis-
tance in constructing several of the clones used in this study. Partial
funding was provided by the New Hampshire Agricultural Experi-
ment Station. This is Scientific Contribution Number 2486.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
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